1. Field
The present disclosure relates to a method for separating a lactone compound having an unsaturated alkyl group, particularly FK506, and lactone compounds having a saturated alkyl group, particularly FK520 and dihydroFK506, as analogs of the lactone compound having an unsaturated alkyl group using a silver ion (Ag+) solution, thereby eliminating the need for column chromatography.
2. Description of the Related Art
FK506 is a tricyclic macrolide lactone compound that is produced by fermentation of Streptomyces species and has immunosuppressive activities. FK506 is used for the prevention of organ transplant rejection and Rh hemolytic diseases of the newborns and the treatment of autoimmune diseases and infectious diseases, etc. FK506 was first reported in 1987 (J. Antibiotics, 16, No. 9, 1249-1255, 1987) and is commercially available from Astellas Pharma. Inc. Since FK506 was first developed, various methods for the separation and purification of FK506 have been proposed in many papers and patents.
Fermentation products obtained by the culture of microorganisms typically contain culture media and a variety of metabolites in the culture solutions. For example, an FK506 fermentation product contains structural analogs of FK506 such as FK520 and dihydroFK506. It is known that FK506 and its structural analogs are mostly produced by microorganisms, particularly microorganisms belonging to Actinomycetes. FK506 was reported to be produced from strains such as Streptomyces tsukubaensis No. 9993, Streptomyces sp. ATCC55098, Streptomyces sp. ATCC53770 and Streptomyces sp. BICC7522 (Muramatsu, H., S. I. Mokhtar, M. Katsuoka and M. Ezaki. 2005, U.S. Pat. No. 4,894,366 and PCT International Publication No. WO 05/098011). FK520 as a structural analog of FK506 also exhibits immunosuppressive activities and antifungal activities and was reported to be produced from Streptomyces hygroscopicus subsp. ascomyceticus ATCC14891, Streptomyces hygroscopicus subsp. yakusimaensis 7238, Streptomyces tsukubaensis 9993, and other species. A fermentation product of an FK506 producing strain contains FK520 and many structures analogous to FK506. It is very important to remove the analogs (e.g., FK520) of FK506 from the fermentation product for the production of high-quality FK506 for medicinal applications.
Under such circumstances, efforts have been made in industrial fields to find methods for removing FK520 and dihydroFK506 to obtain high-purity FK506. These purification techniques are commonly based on column chromatography. It is widely known that silver ion (Ag+) column chromatography is usually used to separate cis- and trans-isomers of unsaturated fatty acids having the same number of carbon atoms (J. chromatography 149 (1978) 417). Silver ion column chromatographic methods for the production of high-purity FK506 are broadly classified into two groups of methods according to the techniques employed: (i) methods using resins pretreated with silver ions (U.S. Pat. No. 6,492,513, U.S. Patent Publication No. 08/000,0834 and PCT International Publication No. WO 05/054253); and (ii) methods for separating a target compound and other compounds by adsorbing a mixture of the compounds to a resin and eluting with a solvent containing silver ions (see U.S. Pat. Nos. 6,576,135 and 6,881,341).
More specifically, U.S. Pat. No. 6,492,513 discloses a method for separating high molecular weight compounds (such as FK506 and FK520) from each other using a sulfonic acid group-containing cation-exchange resin pretreated with silver ions, for example, silver ions provided from silver nitrate.
Further, U.S. Patent Publication No. 08/000,0834 describes a process for the chromatographic separation of FK506 using a silver modified sorbent selected from the group consisting of silver modified aluminum oxide, zirconium oxide, styrene divinylbenzene copolymer, adsorption resin, cation-exchange resin, anion-exchange resin, reverse phase silica gel and cyano silica gel.
Further, PCT International Publication No. WO 05/054253 discloses a process for purifying FK506 including transferring FK506 to an organic solvent using a solvent for layer separation, treating the organic layer with ammonia gas to phase out impurities, and optionally repeating reverse phase chromatography.
The use of expensive resins and silver ions and large amounts of organic solvents in the above-mentioned FK506 purification methods incurs high costs, which are economically disadvantageous. Other problems of the purification methods are much time consumed to perform column chromatography and low purification yields.
U.S. Pat. No. 6,576,135 discloses a method for separating FK506 from a mixture of lactone-containing high molecular weight compounds having one or more alkenyl and alkoxy side chains, the method including adsorbing the mixture to a nonionic adsorption resin and eluting with a solvent containing silver ions, or the method including adsorbing the mixture to active alumina and eluting to remove impurities analogous to FK506 from the lactone-containing high molecular weight compounds.
Although these methods are advantageous in that FK520 and dihydroFK506 can be separated to a large extent from FK506, the use of the high priced resin and a large amount of the silver ion-containing organic solvent causes considerable costs and the removal of silver ions and nitric acid present in the resin is both costly and time consuming, which are economically undesirable.
The foregoing discussion in the background section is to provide general background information, and does not constitute an admission of prior art.